Multi-drug resistant tuberculosis is a major world health problem, especially for HIV infected individuals. There is an urgent need for the development of new tuberculosis drugs that will be active against multi-drug resistant (MDR) and latent tuberculosis infection. The goal of this research is to identify compounds that will block the biosynthesis of mycothiol (MSH). Mycothiol is the major thiol present in actinomycetes and has functions similar to glutathione. MSH biosynthesis has been shown to be essential for growth of Mycobacterium tuberculosis and is hypothesized to be required for its persistence in the dormant state. The goal of this research is to identify compounds that block MSH biosynthesis through development and application of appropriate high-throughput screening (HTS) assays. Such compounds will serve as leads for development of new drugs for MDR TB and will serve as powerful tools for investigation of the biological functions of mycothiol in the many different species of actinomycetes where it is found. The mshA and mshC genes of MSH biosynthesis have been shown to be essential for growth of M. tuberculosis but are not required for growth of M. smegmatis. This makes M. smegmatis a very useful model to identify inhibitors of MSH biosynthesis. In this research, HTS assays will be developed for inhibitors of the two essential enzymes, MshA and MshC, and for inhibitors blocking MSH production in intact M. smegmatis cells. The specific aims of this research are: (1) to develop and apply a HTS assay for inhibitors of MshC, an ATP- dependent ligase, using pyrophosphatase as a coupling enzyme to convert the product pyrophosphate to phosphate which is measured colorimetrically;(2) to develop and apply a whole cell HTS assay with M. smegmatis for inhibitors of MSH production based upon use of monochlorobimane to fluorescently label mycothiol in cells;(3) to develop a HTS assay for inhibitors of MshA, a glycosyltransferase, using an inositol monophosphatase to release phosphate from the product of the MshA reaction, the phosphate being determined colorimetrically;(4) to develop a whole cell HTS assay with M. smegmatis for inhibitors of MSH production based upon growth in the presence of isoniazid by mycothiol-deficient cells and lack of growth of mycothiol-producing cells. These HTS assays are designed to meet the goals of PA-04-068 and will be proposed for screening of libraries available under the Molecular Libraries Initiative.